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• W2039230817 abstract "Integrin-mediated interactions between smooth muscle cells (SMCs) and the extracellular matrix regulate cell migration and proliferation during neointimal hyperplasia. Integrin-linked kinase (ILK) is a serine-threonine kinase and scaffolding molecule that acts downstream of integrin receptors to modulate cell adhesion; therefore, we examined ILK function in SMCs during wound repair. Silencing of ILK expression with siRNA in vitro decreased cell adhesion to fibronectin and accelerated both cell proliferation and wound closure in the cell monolayer; it also resulted in the rearrangement of focal adhesions and diminished central actin stress fibers. Akt and GSK3β are ILK substrates that are important in cell motility; however, ILK siRNA silencing did not attenuate injury-induced increases in Akt and GSK3β phosphorylation. Following balloon catheter injury of the rat carotid artery in vivo, a dramatic decrease in ILK levels coincided with both the proliferation and migration of SMCs, which leads to the formation of a thickened neointima. Immunostaining revealed decreased ILK levels in the media and deep layers of the neointima, but increased ILK levels in the subluminal layers of the intima. Taken together, these results suggest that ILK functions to maintain SMC quiescence in the normal artery. A decrease in ILK levels after injury may permit SMC migration, proliferation, and neointimal thickening, and its re-expression at the luminal surface may attenuate this process during later stages of the injury response. Integrin-mediated interactions between smooth muscle cells (SMCs) and the extracellular matrix regulate cell migration and proliferation during neointimal hyperplasia. Integrin-linked kinase (ILK) is a serine-threonine kinase and scaffolding molecule that acts downstream of integrin receptors to modulate cell adhesion; therefore, we examined ILK function in SMCs during wound repair. Silencing of ILK expression with siRNA in vitro decreased cell adhesion to fibronectin and accelerated both cell proliferation and wound closure in the cell monolayer; it also resulted in the rearrangement of focal adhesions and diminished central actin stress fibers. Akt and GSK3β are ILK substrates that are important in cell motility; however, ILK siRNA silencing did not attenuate injury-induced increases in Akt and GSK3β phosphorylation. Following balloon catheter injury of the rat carotid artery in vivo, a dramatic decrease in ILK levels coincided with both the proliferation and migration of SMCs, which leads to the formation of a thickened neointima. Immunostaining revealed decreased ILK levels in the media and deep layers of the neointima, but increased ILK levels in the subluminal layers of the intima. Taken together, these results suggest that ILK functions to maintain SMC quiescence in the normal artery. A decrease in ILK levels after injury may permit SMC migration, proliferation, and neointimal thickening, and its re-expression at the luminal surface may attenuate this process during later stages of the injury response. Vascular smooth muscle cell (SMC) proliferation, migration, and extracellular matrix synthesis contribute to arterial wound repair and the development of neointimal hyperplasia in important vascular pathologies, including restenosis and atherosclerosis. Interactions between SMCs and the extracellular matrix regulate these processes through binding of the integrin family of cell adhesion receptors. A key mediator of integrin signaling is integrin-linked kinase (ILK), a serine-threonine kinase that interacts with the cytoplasmic domain of β1- and β3-integrin subunits.1Hannigan GE Leung-Hagesteijn C Fitz-Gibbon L Coppolino MG Radeva G Filmus J Bell JC Dedhar S Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase.Nature. 1996; 379: 91-96Crossref PubMed Scopus (972) Google Scholar ILK is also an important scaffolding protein at focal adhesions, where it links integrins to the actin cytoskeleton and regulates actin polymerization.2Wu C The PINCH-ILK-parvin complexes: assembly, functions and regulation.Biochim Biophys Acta. 2004; 1692: 55-62Crossref PubMed Scopus (124) Google Scholar In many cell types, ILK is activated by phosphatidylinositol 3-kinase (PI3K), and regulates the activity of the downstream kinases protein kinase B/Akt (Akt) and glycogen synthase kinase 3 β (GSK3β) through phosphorylation.3Persad S Attwell S Gray V Mawji N Deng JT Leung D Yan J Sanghera J Walsh MP Dedhar S Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343.J Biol Chem. 2001; 276: 27462-27469Crossref PubMed Scopus (417) Google Scholar, 4Delcommenne M Tan C Gray V Rue L Woodgett J Dedhar S Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase.Proc Natl Acad Sci USA. 1998; 95: 11211-11216Crossref PubMed Scopus (950) Google Scholar ILK has been implicated in processes involving cell proliferation and motility, including anchorage-independent growth, survival, and cell cycle progression,5Persad S Dedhar S The role of integrin-linked kinase (ILK) in cancer progression.Cancer Metastasis Rev. 2003; 22: 375-384Crossref PubMed Scopus (148) Google Scholar, 6Fielding AB Dobreva I McDonald PC Foster LJ Dedhar S Integrin-linked kinase localizes to the centrosome and regulates mitotic spindle organization.J Cell Biol. 2008; 180: 681-689Crossref PubMed Scopus (106) Google Scholar as well as the epithelial-mesenchymal transition, invasion, and migration.7Oloumi A McPhee T Dedhar S Regulation of E-cadherin expression and beta-catenin/Tcf transcriptional activity by the integrin-linked kinase.Biochim Biophys Acta. 2004; 1691: 1-15Crossref PubMed Scopus (159) Google Scholar Evidence from genetic mouse models demonstrates important roles for ILK in vascular development8Friedrich EB Liu E Sinha S Cook S Milstone DS MacRae CA Mariotti M Kuhlencordt PJ Force T Rosenzweig A St-Arnaud R Dedhar S Gerszten RE Integrin-linked kinase regulates endothelial cell survival and vascular development.Mol Cell Biol. 2004; 24: 8134-8144Crossref PubMed Scopus (119) Google Scholar, 9Vouret-Craviari V Boulter E Grall D Matthews C Van Obberghen-Schilling E ILK is required for the assembly of matrix-forming adhesions and capillary morphogenesis in endothelial cells.J Cell Sci. 2004; 117: 4559-4569Crossref PubMed Scopus (69) Google Scholar tumor angiogenesis,10Kaneko Y Kitazato K Basaki Y Integrin-linked kinase regulates vascular morphogenesis induced by vascular endothelial growth factor.J Cell Sci. 2004; 117: 407-415Crossref PubMed Scopus (48) Google Scholar, 11Tan C Cruet-Hennequart S Troussard A Fazli L Costello P Sutton K Wheeler J Gleave M Sanghera J Dedhar S Regulation of tumor angiogenesis by integrin-linked kinase (ILK).Cancer Cell. 2004; 5: 79-90Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar and cardiac hypertrophy.12White DE Coutu P Shi YF Tardif JC Nattel S St AR Dedhar S Muller WJ Targeted ablation of ILK from the murine heart results in dilated cardiomyopathy and spontaneous heart failure.Genes Dev. 2006; 20: 2355-2360Crossref PubMed Scopus (139) Google Scholar, 13Lu H Fedak PWM Dai X Du C Zhou YQ Henkelman M Mongroo PS Lau A Yamabi H Hinek A Husain M Hannigan G Coles JG Integrin-linked kinase expression is elevated in human cardiac hypertrophy and induces hypertrophy in transgenic mice.Circulation. 2006; 114: 2271-2279Crossref PubMed Scopus (108) Google Scholar, 14Hannigan GE Coles JG Dedhar S Integrin-linked kinase at the heart of cardiac contractility, repair, and disease.Circ Res. 2007; 100: 1408-1414Crossref PubMed Scopus (134) Google Scholar In SMCs, ILK phosphorylates myosin light chain and myosin phosphatase and may therefore influence cytoskeletal function and cell contraction.15Deng JT Van Lierop JE Sutherland C Walsh MP Ca2+-independent smooth muscle contraction. a novel function for integrin-linked kinase.J Biol Chem. 2001; 276: 16365-16373Crossref PubMed Scopus (223) Google Scholar, 16Wilson DP Sutherland C Borman MA Deng JT Macdonald JA Walsh MP Integrin-linked kinase is responsible for Ca2+-independent myosin diphosphorylation and contraction of vascular smooth muscle.Biochem J. 2005; 392: 641-648Crossref PubMed Scopus (94) Google Scholar, 17Muranyi A Macdonald JA Deng JT Wilson DP Haystead TA Walsh MP Erdodi F Kiss E Wu Y Hartshorne DJ Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase.Biochem J. 2002; 366: 211-216Crossref PubMed Google Scholar Despite this research, little is known about ILK in arterial wound repair and intimal hyperplasia. In this study, we used knockdown strategies to examine ILK function following SMC injury and wound repair in vitro. ILK expression and activity were measured following balloon catheter injury of the rat carotid artery, a model used to study SMC responses to injury. Polyclonal antibodies from Cell Signaling Technology included anti-ILK antibody (#3862), anti-phospho-Akt(Ser473) (#9271), anti-Akt (#9272), and anti-phospho-GSK3β(Ser9) (#9336). The monoclonal anti-GSK3β antibody (clone 4G-1E), and the polyclonal anti-ILK antibody (#06-592) used for immunoprecipitation were from Upstate Cell Signaling Solutions. Rabbit polyclonal anti-β-actin antibody (ab8227) was from Abcam, and the mouse monoclonal anti-α-actin antibody (clone asm-1) was from Boehringer Mannheim Biochemica. To examine the role of ILK in the SMC response to injury, we performed a scrape wound assay on confluent SMCs grown in vitro, an assay that mimics the cell migration and proliferation that occurs during intimal hyperplasia. Primary medial SMCs were obtained from the rat carotid artery, and propagated in culture as previously described.18Franco CD Ho B Mulholland D Hou G Donaldson KL Bendeck MP Doxycycline alters vascular smooth muscle cell adhesion, migration and reorganization of fibrillar collagen matrices.Am J Pathol. 2006; 168: 1697-1709Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Cells were used from passages 4 to 10. Cells were grown to confluence in a 6-well tissue culture plate, then wounded with a P20 pipette tip to create a lengthwise wound measuring ∼300 um in width, and incubated in standard growth media, Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 2% penicillin-streptomycin. Measurements of wound width were obtained every 12 hours over a period of 60 hours using Simple PCI Imaging software (C-Imaging, Mars, PA). Mean values were expressed as a percent wound closure, which was calculated as follows: Percent wound closure = 1 − (widtht/width0) × 100%. These experiments were performed in duplicate and repeated three times. To determine the importance of ILK in regulating wound closure, ILK expression in SMCs was attenuated by RNA interference. SMCs were transfected in suspension with either a non-targeting control siRNA (Dharmacon siCONTROL) as a control for non-sequence-specific effects, or an ILK-specific siRNA sequence (Silencer Pre-designed siRNA ID #56983 from Ambion, Austin TX). The ILK siRNA targets sequence in exon 8 of the rat mRNA for ILK (NM_133409). For each well, 90 pmol of either the non-targeting or ILK-specific duplex was diluted in 500 μl Opti-MEM I reduced serum medium and 5 μl of Lipofectamine RNAiMAX was added. The mixture was gently mixed and allowed to incubate for 20 minutes at room temperature. SMCs grown in T75 tissue culture flasks were trypsinized, spun down, and resuspended in standard growth medium without antibiotics such that 2.5 ml media contained 200,000 cells. To each well containing RNAi duplex-Lipofectamine RNAiMAX complexes, 200,000 cells were added, to give a final RNA concentration of 30 nmol/L. Twenty-four hours after transfection, antibiotic-free medium was replaced with standard growth medium. Once the cells reached confluence, they were wounded. Attenuation of ILK expression was verified by Western blotting of cell lysates probed with antibody against ILK. To measure cell proliferation, SMCs were transfected and plated in 6-well plates as described above. Twenty-four hours after transfection, the antibiotic-free medium was replaced with media containing antibiotics and 0.5% serum to starve the cells for 16 hours. The cells were then wounded using a comb with multiple teeth each 1 mm wide, and media was replaced with standard growth medium containing 20 μl/ml Alamar Blue (Biosource, Camarillo, CA 93012) and incubated for 1 hour. Media was removed and the fluorescence was read at excitation 560 nm and emission 590 nm using a Fluostar Optima fluorescence reader (BMG Lab Technologies). Cells were replenished with fresh media, and the Alamar Blue procedure was repeated at 24 and 48 hours after injury. Blank values read from wells containing no cells were obtained at each time point and were subtracted, and the ratio of the fluorescence measured at 24 or 48 hours relative to the measurement at 1 hour was calculated. Wells were assayed in triplicate, and the entire experiment was repeated three times. Attachment assays were performed in 96-well plates precoated with 10 nmol/L fibronectin as previously described.19Hou G Vogel W Bendeck MP The discoidin domain-receptor tyrosine kinase DDR1 in arterial wound repair.J Clin Invest. 2001; 107: 727-735Crossref PubMed Scopus (186) Google Scholar SMCs were treated with either non-targeting or ILK siRNA as described above, then 60,000 cells were added to each well, and the cells were allowed to attach for 90 minutes. Nonadherent cells were washed off with PBS, the remaining cells were stained with 0.5% toluidine blue, and the absorbance of the solution in the well was measured at 595 nm in a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). The experiment was done in duplicate and repeated three times. Following incubation with either control non-targeting siRNA or ILK siRNA, subconfluent layers of SMCs were plated on 35-mm dishes. Oregon-green-labeled human plasma fibronectin (provided by G. Pickering20Pickering JG Chow LH Li S Rogers KA Rocnik EF Zhong R Chan BMC {alpha}5{beta}1 integrin expression and luminal edge fibronectin matrix assembly by smooth muscle cells after arterial injury.Am J Pathol. 2000; 156: 453-465Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) was added to the cells at a concentration of 100 nmol/L in standard growth medium, and the cells were incubated for 24 hours. The cells were washed with PBS to remove non-assembled fibronectin, then fixed with 4% paraformaldehyde for 15 minutes, and stained with Hoescht 33258 for 30 minutes to label cell nuclei. This experiment was repeated twice. Subconfluent cultures of rat SMCs were stained with antibodies against ILK and paxillin to determine the localization of ILK and focal adhesion complexes. Cells were transfected with control siRNA or ILK siRNA as described above, then plated on glass coverslips in 24-well plates; 24 hours after transfection the antibiotic-free medium was replaced with standard growth medium, and the cells were incubated for an additional 18 hours. Cells were washed with PBS then fixed with methanol and 4% paraformaldehyde at a 1:1 dilution for 15 minutes. The cells were stained with anti-ILK and anti-paxillin (BD Bioscience #650051) antibodies at dilutions of 1:100 for 16 hours at 4°C. Cells were then incubated with secondary antibodies [CY3-conjugated anti-rabbit antibody and fluorescein isothiocyanate-conjugated anti-mouse antibody, both at 1:200 in PBS], and with Hoescht nuclear stain (Hoescht 33258, Sigma, Saint Louis, MO) diluted 1:1000 for 30 minutes. Cells were also double stained with ILK antibody and tetramethylrhodamine isothiocyanate-phalloidin to stain actin filaments. For these experiments, cells were fixed in 4% paraformaldehyde and methanol at a ratio of 2:1 for 15 minutes. The ILK antibody was applied as described above, and tetramethylrhodamine isothiocyanate-phalloidin (Sigma #P1951) was applied at a 1:500 dilution. The secondary antibody was a fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (Jackson Immunoresearch #111-095-045). Cells were visualized using an Olympus Fluoview confocal microscope. SMCs were grown to confluence in 60-mm tissue culture dishes, then wounded along perpendicular axes using a sterilized comb (37-mm wide with 13 × 1-mm teeth) to create a grid wound. After wounding, fresh media was added and SMCs were allowed to proliferate and migrate for periods of 5 minutes, 30 minutes, and 1, 2, 4, 8, and 24 hours. Cells were lysed in 500 μl lysis buffer, and the lysates were used for Western blotting to measure phospho-Akt, Akt, phospho-GSK3β, GSK3β, and β-actin. siRNA was also used to determine whether ILK mediated the phosphorylation of Akt and GSK3β in the wounded SMC cultures. In 60-mm culture dishes, 180 pmol of either the non-targeting or ILK-specific duplex was diluted in 1 ml Opti-MEM I reduced serum medium. Ten μl of Lipofectamine RNAiMAX was then added to each dish. The mixture was gently mixed and allowed to incubate for 20 minutes at room temperature. SMCs grown in T75 tissue culture flasks were trypsinized, spun down, and resuspended in standard growth medium without antibiotics, such that 5 ml media contained 400,000 cells. To each dish containing RNAi duplex-Lipofectamine RNAiMAX complexes, 400,000 cells were added yielding a final RNA concentration of 30 nmol/L. The antibiotic-free medium was replaced with standard growth medium, 24 hours after transfection. Cells were grown to confluence and were then subject to grid wounding, 72 hours after transfection. Cells were lysed at various time points after injury, and the lysates were subject to Western blotting and probed with antibodies against phospho-Akt, phospho-GSK3β, and β-actin. Western blots containing cell lysates (10 μg total protein per lane) were incubated for 16 hours at 4°C with anti-ILK, anti-phospho-Akt, anti-Akt, anti-phospho-GSK3β, or anti-GSK3 antibodies diluted 1:1000 in blocking buffer. Secondary antibodies used were horseradish peroxidase-conjugated donkey anti-rabbit or sheep anti-mouse antibodies at a dilution of 1:2000. Detection was performed using enhanced chemiluminescence (PerkinElmer Inc., Boston, MA). Measurements of band density were performed using Image J for Windows software (National Institutes of Health). ILK, phospho-Akt and phospho-GSK3β were normalized to β-actin, total Akt and GSK3β, respectively, and then further normalized to a single sample loaded on all immunoblots. Each experiment was repeated three to four times. Male Sprague-Dawley rats (Charles River, Constant, QB), 3 to 4 months old and weighing 350 to 400g were used. Animal experiments were performed in accordance with the guidelines of the Canada Council on Animal Care. Rats were anesthetized by intraperitoneal injection of xylazine (4.6 mg/kg body weight, Rompum; Bayer Inc., Etobicoke, ON) and ketamine (70 mg/kg body weight, Ketaset; Ayerst Veterinarian Laboratories, Guelph, ON). Balloon catheter injury of the left carotid artery was performed as previously described.21Bendeck MP Conte M Zhang M Nili N Strauss BH Farwell SM Doxycycline modulates smooth muscle cell growth, migration and matrix remodeling after arterial injury.Am J Pathol. 2002; 160: 1089-1095Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar Rats were sacrificed by an overdose of ketamine (350 mg/kg), the common carotid arteries were cleared in situ, and the central portion 20 mm in length was removed and snap-frozen in liquid nitrogen. Each artery was pulverized in liquid nitrogen and the powder was collected in 100 μl lysis buffer, composed of 34.7 M SDS, 1 mmol/L phenylmethylsulfonyl fluoride, and 23.4 μmol/L leupeptin in 44.5 mmol/L Tris pH 7.6. Samples were centrifuged at 15,000 relative centrifugal force for 5 minutes at room temperature. Total protein content in the supernatant was measured using the BioRad Detergent-Compatible Microplate Assay. The remainder of the supernatant was diluted 1:1 with 2× sample buffer composed of 0.1 M Tris pH 6.8, 69.4 mmol/L SDS, 20% glycerol and 0.1% bromphenol blue. Native SDS-polyacrylamide gels were loaded with 10 μg of protein per lane, and subject to electrophoresis followed by transfer of proteins from the gel to polyvinylidene difluoride membranes. The western bots were incubated for 16 hours at 4°C with anti-ILK antibodies diluted 1:1000 in blocking buffer. Secondary antibodies were conjugated with horseradish peroxidase, and detection was performed using enhanced chemiluminescence exposing autoradiographs. Blots were stripped and reprobed with antibody against Akt to ensure even loading of proteins in the lanes, and were finally stained with Ponceau to confirm even protein loading. Measurements of band density on the autoradiographs were made using Image J for Windows. All values were expressed relative the uninjured control carotid value on the same blot. These analyses included at least four rats per time point; thus the values presented represent the mean ± SEM from four animals. Fourteen days after balloon catheter injury, arteries were excised and cleared. The arteries were cut open longitudinally and pinned lumen side up onto silicone-coated plates. The intima was harvested by scoring the vessel at one end, then grasping the edge of the intima with forceps placed across the width of the vessel and peeling back the intima. The intima and media of each artery was lysed separately in 50 μl lysis buffer. Protein was quantified as described above, and ILK expression in intimal and medial lysates was assessed by western blotting. These analyses included at least three rats. An assay to measure ILK activity was performed using myelin basic protein (MBP) as a substrate for phosphorylation by ILK. Uninjured rat common carotid arteries, and arteries harvested 14 days after balloon catheter injury were lysed in 100 μl Nonidet P-40 (NP-40) lysis buffer composed of 145 mmol/L sodium chloride, 1% NP-40, 11.7 mmol/L sodium deoxycholate, 48.4 mmol/L Hepes pH 7.5, 2.34 μmol/L leupeptin, 154 nmol/L aprotinin, and 3 mmol/L phenylmethylsulfonyl fluoride. ILK was immunoprecipitated from 160 μg of total protein from arterial lysates by incubating with 1.44 μg rabbit polyclonal anti-ILK antibody (Upstate Cell Signaling) at 4°C for 16 hours. Immune complexes were collected in 50 μl of 50% protein A agarose in NP40 lysis buffer, rotated at 4°C for 2 hours, then washed with NP40 lysis buffer, and then kinase wash buffer (10 mmol/L MgCl2, 10 mmol/L MnCl2, 50 mmol/L Hepes pH 7.5, 0.1 mmol/L sodium orthovanadate, 1 mmol/L dithiothreitol). Twenty-five μl radioactive reaction buffer (10 mmol/L MgCl2, 10 mmol/L MnCl2, 50 mmol/L Hepes pH 7.5, 1 mmol/L sodium orthovanadate, 2 mmol/L sodium fluoride) was added to the reaction and combined with 5 μg MBP and 5 μCi [γ32P] ATP. After a 30-minute incubation at 37°C, the reaction was terminated with 8 μl of 5× SDS sample buffer; 35 μl of reaction volume was loaded into each well and separated on 12% SDS-polyacrylamide electrophoresis gels. The gels were dried and phosphorylation of MBP was detected by autoradiography. ILK kinase activity was normalized to total ILK protein expression as assessed on a Western blot prepared using total arterial lysates of the same arteries. Measurements of band density in autoradiographs and Western blots were performed using Image J for Windows software. The band density in autoradiographs was divided by the band density of the corresponding sample in Western blots, yielding a measure of ILK kinase activity per unit of ILK protein. Experiments included at least four rats. Rats were subject to balloon catheter injury of the rat carotid artery then sacrificed at 2, 7, or 14 days after injury. Immediately after sacrifice of the rat the carotid arteries were perfusion-fixed with 4% paraformaldehyde at constant physiological pressure (110 mmHg) for 10 minutes. Arteries were embedded in paraffin and cut into 5 μm-thick cross-sections. Sections were deparaffinized in xylenes, rehydrated in a gradient of anhydrous ethanol and rinsed with PBS. For antigen retrieval, sections were immersed in 10 mmol/L sodium citrate (pH 6.0) and boiled for 10 minutes in a microwave set at high. Sections were stained for 16 hours at 4°C with polyclonal anti-ILK antibody in 1% normal goat serum. Sections were then incubated with biotinylated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories) at 1:400 for 1 hour at room temperature. Avidin-biotin-peroxidase reagent (Vectastain Elite ABC Kit; Vector Laboratories), followed by 3,3′-diaminobenzidine (DAB Substrate Kit for Peroxidase; Vector Laboratories) was used for detection. Statistical analysis was performed using SigmaStat software (Systat Software Inc., San Jose, CA). Group means were compared by analysis of variance followed by Tukey's post hoc test for pairwise comparisons. For the SMC wound closure assay, a repeated measures analysis of variance was performed to compare the group means at each time point. For the cell attachment, cell proliferation, and fibronectin assembly assays, means were compared by student's t-test. Statistical significance was set at P < 0.05. Western blots showed nearly complete inhibition of ILK protein expression after cells were transfected with targeted siRNA (Figure 1A), and there was no evidence for knockdown of unintended targets (eg, β-actin, Akt or GSK-3β) (refer to Figure 3C, below; data not shown). Wound closure was significantly accelerated in ILK-silenced cells compared with untransfected or control siRNA transfected cells at 24 hours after wounding (Figure 1B). However the wounds closed in all groups by 60 hours, suggesting that the presence of ILK delayed but did not prevent wound closure. Since wound closure is the result of both migration and proliferation of SMCs, we also studied the effect of ILK-silencing on cell proliferation. Cell proliferation was increased by 1.8-fold in ILK-silenced cells compared with control siRNA transfected cells at 24 hours (P < 0.017). Taken together, these data suggest that increased proliferation along with increased cell migration contributed to the acceleration of wound closure.Figure 3Wounding SMCs results in transient increases in phospho-Akt and phospho-GSK3β that are not affected by ILK silencing. A: Representative immunoblots of lysates from unwounded (UI) and wounded SMCs after injury, probed with antibodies against phospho-Akt or total Akt. Densitometric analysis of phospho-Akt expression revealed a significant increase at 5 minutes after wounding. Values were normalized to total Akt to control for loading, and expressed relative to the uninjured control. B: Representative immunoblots of cell lysates from unwounded (UI) and wounded SMCs probed for phospho-GSK3β and total GSK3β. Densitometric analysis revealed a significant increase at 5 minutes after injury. Values were normalized to total GSK3β to control for loading, and expressed relative to the uninjured control. *P < 0.05 compared with unwounded cells. C: Phosphorylation of Akt and GSK3β is not dependent on ILK in wounded SMCs. Top panel: Western blot containing cell lysates, and probed for ILK, shows effective down-regulation of ILK expression by siRNA; second and third panels show Western blots probed for phospho-Akt and phospho-GSK3β, and reveal increases in phosphorylation after wounding. Silencing ILK did not inhibit the phosphorylation of either mediator. Blots were stripped and reprobed with β-actin to demonstrate equal loading. The numbers underneath each panel represent the band density in that lane expressed as a fold change relative to the band in uninjured, untransfected cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Tight control of adhesive forces is necessary for optimum cell migration; accordingly, strong adhesion can prevent or delay migration.22Palecek SP Loftus JC Ginsberg MH Lauffenburger DA Horwitz AF Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness.Nature. 1997; 385: 537-540Crossref PubMed Scopus (1198) Google Scholar We assayed the adhesion of control and ILK-silenced SMCs to fibronectin-coated plates. Silencing ILK expression significantly decreased cell attachment by 33% (Figure 1C). Because ILK mediates fibronectin fibrillogenesis in fibroblasts,23Wu C Keightley SY Leung-Hagesteijn C Radeva G Coppolino M Goicoechea S McDonald JA Dedhar S Integrin-linked protein kinase regulates fibronectin matrix assembly. E-cadherin expression, and tumorigenicity.J Biol Chem. 1998; 273: 528-536Crossref PubMed Scopus (252) Google Scholar, 24Kagami S Shimizu M Kondo S Kitamura A Urushihara M Takamatsu M Yamaji S Ishigatsubo Y Kawachi H Shimizu F Up-regulation of integrin-linked kinase activity in rat mesangioproliferative glomerulonephritis.Life Sci. 2006; 78: 1794-1800Crossref PubMed Scopus (13) Google Scholar as further evidence of ILK functional knockdown, we measured fibrillogenesis in SMCs by incubating Oregon-green-labeled fibronectin protomers on a layer of SMCs in vitro. Silencing ILK decreased the assembly of fibronectin fibrils on the SMC surface (Figure 1D). Results were quantified by image analysis, and the fraction of the microscopic field occupied by fibronectin fibrils was reduced from 11.15 ± 1.89% in control cultures, to 4.37 ± 0.86% after siRNA treatment to reduce ILK (∼60% reduction, P < 0.05). ILK can act as a scaffolding protein at focal adhesion sites, linking integrins to the actin cytoskeleton, which could impact on cell migration, therefore" @default.
• W2039230817 created "2016-06-24" @default.
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• W2039230817 date "2008-07-01" @default.
• W2039230817 modified "2023-10-16" @default.
• W2039230817 title "Integrin-Linked Kinase in the Vascular Smooth Muscle Cell Response to Injury" @default.
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• W2039230817 doi "https://doi.org/10.2353/ajpath.2008.071046" @default.
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